Investigating the inhibitory effect of miR-34a, miR-449a, miR-1827, and miR-106b on target genes including NOTCH1, c-Myc, and CCND1 in human T cell acute lymphoblastic leukemia clinical samples and cell line

Document Type: Original Article

Authors

1 Department of laboratory hematology and blood bank, School of allied medicine, Shahid Beheshti University of medical sciences, Tehran, Iran

2 Medical nanotechnology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran

3 Department of Biotechnology, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran

4 Department of Biochemistry, School of Medicine, Semnan University of Medical Sciences, Semnan, Iran

5 Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran

6 Mahak charity hospital, Tehran, Iran

7 Department of Research and Development, Production and Research Complex, Pasteur Institute of Iran, Tehran, Iran

Abstract

Objective(s): microRNAs are small non-coding molecules that regulate gene expression in various biological processes. T-cell acute lymphoblastic leukemia (T-ALL) is a malignancy accompanied with genetic aberrations and accounts for 20% of children’s and adult’s ALL. Notch signaling pathway dysregulation occurs in 60% of T-ALL cases. In the present study, we aimed to determine the relationship between miRNAs and genes involved in Notch signaling pathway.
Materials and Methods: Considering the role of the pathway and its down-stream genes in proliferation, differentiation, cell cycle, and apoptosis, NOTCH1, c-Myc, and CCND1 genes were selected as target genes. Using bioinformatics studies, miR-34a, miR-449a, miR-1827, and miR-106b were selected as miRNAs targeting the above-mentioned genes. We evaluated these genes and miRNAs in T-ALL clinical samples as well as Jurkat cell line, in which NOTCH1 is overexpressed.
Results: Quantitative Real-Time PCR indicated that NOTCH1, c-Myc, and CCND1 were overexpressed in samples with decreased expression of miR-34a. In addition, we observed that samples with decreased expression of miR-449a showed increased expression of NOTCH1 and CCND1. Furthermore, we analyzed the expression of miR-1827 and miR-106b, which target c-Myc and CCND1, respectively. We found out that the expression of miR-1827, miR-106b, and their respective target genes were inversely correlated in 80% and 75% of the cases (r=0.8), respectively. Furthermore, in Jurkat cell line, the expression of target genes was increased while the candidate miRNAs except miR-34a were decreased.
Conclusion: These miRNAs can be proposed as biomarkers and new therapeutic targets in T-ALL patients who have NOTCH1 overexpression.

Keywords


1. Wang Z, Li Y, Ahmad A, Azmi AS, Banerjee S, Kong D, et al. Targeting Notch signaling pathway to overcome drug resistance for cancer therapy. Biochim Biophys Acta Rev Cancer. 2010;1806:258-267.
2. Sarmento L, Póvoa V, Nascimento R, Real G, Antunes I, Martins L, et al. CHK1 overexpression in T-cell acute lymphoblastic leukemia is essential for proliferation and survival by preventing excessive replication stress. Oncogene. 2015;34:2978-2990.
3. Lobry C, Oh P, Mansour MR, Look AT, Aifantis I. Notch signaling: switching an oncogene to a tumor suppressor. Blood. 2014;123:2451-2459.
4. Hasserjian R, Aster J, Davi F, Weinberg D, Sklar J. Modulated expression of notch1 during thymocyte development. Blood. 1996;88:970-976.
5. Liu J, Sato C, Cerletti M, Wagers A. Chapter twelve-notch signaling in the regulation of stem cell self-renewal and differentiation. Curr Top Dev Biol. 2010;92:367-409.
6. Deftos ML, He Y-W, Ojala EW, Bevan MJ. Correlating notch signaling with thymocyte maturation. Immunity. 1998;9:777-786.
7. van Limpt V, Chan A, Caron H, Sluis PV, Boon K, Hermus MC, et al. SAGE analysis of neuroblastoma reveals a high expression of the human homologue of the Drosophila Delta gene. Med Pediatr Oncol. 2000;35:554-558.
8. Su Q, Xin L. Notch signaling in prostate cancer: refining a therapeutic opportunity. Histol Histopathol. 2016;31:149-157.
9. Stylianou S, Clarke RB, Brennan K. Aberrant activation of notch signaling in human breast cancer. Cancer Res. 2006;66:1517-1525.
10. Stockhausen MT, Kristoffersen K, Poulsen HS. The functional role of Notch signaling in human gliomas. Neuro Oncol. 2010;12:199-211.
11. Ye Q, Jiang J, Zhan G, Yan W, Huang L, Hu Y, et al. Small molecule activation of NOTCH signaling inhibits acute myeloid leukemia. Sci Rep. 2016;6:26510.
12. Malecki MJ, Sanchez-Irizarry C, Mitchell JL, Histen G, Xu ML, Aster JC, et al. Leukemia-associated mutations within the NOTCH1 heterodimerization domain fall into at least two distinct mechanistic classes. Mol Cell Biol. 2006;26:4642-4651.
13. Joshi I, Minter LM, Telfer J, Demarest RM, Capobianco AJ, Aster JC, et al. Notch signaling mediates G 1/S cell-cycle progression in T cells via cyclin D3 and its dependent kinases. Blood. 2009;113:1689-1698.
14. Gautschi O, Ratschiller D, Gugger M, Betticher DC, Heighway J. Cyclin D1 in non-small cell lung cancer: a key driver of malignant transformation. Lung cancer. 2007;55:1-14.
15. Yang CC, Chu KC, Chen HY, Chen WC. Expression of p16 and cyclin D1 in bladder cancer and correlation in cancer progression. Urol Int. 2002;69:190-194.
16. Bachmann K, Neumann A, Hinsch A, Nentwich MF, El Gammal AT, Vashist Y, et al. Cyclin D1 is a strong prognostic factor for survival in pancreatic cancer: analysis of CD G870A polymorphism, FISH and immunohistochemistry. J Surg Oncol. 2015;111:316-323.
17. Cohen B, Shimizu M, Izrailit J, Ng NF, Buchman Y, Pan JG, et al. Cyclin D1 is a direct target of JAG1-mediated Notch signaling in breast cancer. Breast Cancer Res Treat. 2010;123:113-124.
18. Ghildiyal M, Zamore PD. Small silencing RNAs: an expanding universe. Nat Rev Genet. 2009;10:94-108.
19. Winter J, Jung S, Keller S, Gregory RI, Diederichs S. Many roads to maturity: microRNA biogenesis pathways and their regulation. Nat Cell Biol. 2009;11:228-234.
20. Wang W, Luo Y-p. MicroRNAs in breast cancer: oncogene and tumor suppressors with clinical potential. J Zhejiang Univ Sci B. 2015;16:18-31.
21. Abba ML, Patil N, Leupold JH, Moniuszko M, Utikal J, Niklinski J, et al. MicroRNAs as novel targets and tools in cancer therapy. Cancer lett. 2017;387:84-94.
22. Karami F, Mohammadi-Yeganeh S, Abedi N, Koochaki A, Kia V, Paryan M. Bioinformatics Prediction and In Vitro Analysis Revealed That miR-17 Targets Cyclin D1 mRNA in Triple Negative Breast Cancer Cells. Chem Biol Drug Des. 2016;87:317-320.
23. Mohammadi-Yeganeh S, Mansouri A, Paryan M. Targeting of miR9/NOTCH1 interaction reduces metastatic behavior in triple-negative breast cancer. Chem Biol Drug Des. 2015;86:1185-1191.
24. Mohammadi-Yeganeh S, Paryan M, Samiee SM, Soleimani M, Arefian E, Azadmanesh K, et al. Development of a robust, low cost stem-loop real-time quantification PCR technique for miRNA expression analysis. Mol Biol Rep. 2013;40:3665-3674.
25. Paryan M, Mohammadi-Yeganeh S, Samiee SM, Soleimani M, Arefian E, Azadmanesh K, et al. Investigation of deregulated genes of Notch signaling pathway in human T cell acute lymphoblastic leukemia cell lines and clinical samples. Mol Biol Rep. 2013;40:5531-5540.
26. Li X, von Boehmer H. Notch signaling in T-cell development and T-ALL. ISRN Hematol. 2011;2011.
27. Xu Y, Yang J, Li X. MicroRNA-25 promotes T-cell acute lymphoblastic leukemia cell proliferation and invasion by directly targeting EphA8. IJCEP. 2016;9:5292-5298.
28. Shi C, Zhang X, Li X, Zhang L, Li L, Sun Z, et al. Effects of microRNA-21 on the biological functions of T-cell acute lymphoblastic lymphoma/leukemia. Oncol Lett. 2016;12:4173-4180.
29. Cimmino A, Calin GA, Fabbri M, Iorio MV, Ferracin M, Shimizu M, et al. miR-15 and miR-16 induce apoptosis by targeting BCL2. Proc Natl Acad Sci U S A. 2005;102:13944-13949.
30. Wang J, Chen J, Sen S. MicroRNA as biomarkers and diagnostics. J Cell Physiol. 2016;231:25-30.
31. Mavrakis KJ, Van Der Meulen J, Wolfe AL, Liu X, Mets E, Taghon T, et al. A cooperative microRNA-tumor suppressor gene network in acute T-cell lymphoblastic leukemia (T-ALL). Nat Genet. 2011;43:673-678.
32. Lv M, Zhang X, Jia H, Li D, Zhang B, Zhang H, et al. An oncogenic role of miR-142-3p in human T-cell acute lymphoblastic leukemia (T-ALL) by targeting glucocorticoid receptor-[alpha] and cAMP/PKA pathways. Leukemia. 2012;26:769-777.
33. Gusscott S, Kuchenbauer F, Humphries RK, Weng AP. Notch-mediated repression of miR-223 contributes to IGF1R regulation in T-ALL. Leuk Res. 2012;36:905-911.
34. Li X, Sanda T, Look AT, Novina CD, von Boehmer H. Repression of tumor suppressor miR-451 is essential for NOTCH1-induced oncogenesis in T-ALL. J Exp Med. 2011:663-675.
35. Mohammadi Yeganeh S, Vasei M, Tavakoli R, Kia V, Paryan M. The effect of miR-340 over-expression on cell-cycle-related genes in triple-negative breast cancer cells. Eur J Cancer Care (Engl). 2017;26.
36. Weng AP, Millholland JM, Yashiro-Ohtani Y, Arcangeli ML, Lau A, Wai C, et al. c-Myc is an important direct target of Notch1 in T-cell acute lymphoblastic leukemia/lymphoma Genes Dev. 2006;20:2096-2109.
37. Palomero T, Lim WK, Odom DT, Sulis ML, Real PJ, Margolin A, et al. NOTCH1 directly regulates c-MYC and activates a feed-forward-loop transcriptional network promoting leukemic cell growth. Proc Natl Acad Sci U S A. 2006;103:18261-18266.
38. Tao J, Zhao X, Tao J. c-MYC–miRNA circuitry: a central regulator of aggressive B-cell malignancies. Cell Cycle. 2014;13:191-198.
39. Christoffersen N, Shalgi R, Frankel L, Leucci E, Lees M, Klausen M, et al. p53-independent upregulation of miR-34a during oncogene-induced senescence represses MYC. Cell death differ. 2010;17:236-245.
40. Sun F, Fu H, Liu Q, Tie Y, Zhu J, Xing R, et al. Downregulation of CCND1 and CDK6 by miR‐34a induces cell cycle arrest. FEBS Lett. 2008;582:1564-1568.
41. Du R, Sun W, Xia L, Zhao A, Yu Y, Zhao L, et al. Hypoxia-induced down-regulation of microRNA-34a promotes EMT by targeting the Notch signaling pathway in tubular epithelial cells. PloS one. 2012;7:e30771.
42. De Weer A, Van der Meulen J, Rondou P, Taghon T, Konrad TA, De Preter K, et al. EVI1‐mediated down regulation of MIR449A is essential for the survival of EVI1 positive leukaemic cells. Br J Haematol. 2011;154:337-348.
43. Capuano M, Iaffaldano L, Tinto N, Montanaro D, Capobianco V, Izzo V, et al. MicroRNA-449a overexpression, reduced NOTCH1 signals and scarce goblet cells characterize the small intestine of celiac patients. PloS one. 2011;6:e29094.
44. Shi J, Liu Y, Liu J, Zhou J. Hsa-miR-449a genetic variant is associated with risk of gastric cancer in a Chinese population. Int J Clin Exp Pathol. 2015;8:13387-13392.
45. Zhang C, Liu J, Tan C, Yue X, Zhao Y, Peng J, et al. microRNA-1827 represses MDM2 to positively regulate tumor suppressor p53 and suppress tumorigenesis. Oncotarget. 2016;7:8783-8796.
46. Lin N, Zhou Y, Lian X, Tu Y. Expression of microRNA-106b and its clinical significance in cutaneous melanoma. Genet Mol Res. 2015;14:16379-16385.
47. Jiang L, Li X, Cheng Q, Zhang B-H. Plasma microRNA might as a potential biomarker for hepatocellular carcinoma and chronic liver disease screening. Tumour Biol. 2015;36:7167-7174.

sion
In the present study, considering the role of Notch signaling pathway and miRNAs in T-ALL, we aimed to evaluate the most specific genes of Notch pathway and their specific targeting miRNAs. By analyzing data obtained from bioinformatics methods and databases, an inverse correlation between NOTCH1, c-Myc, and CCND1 and their targeting miRNAs was considered to be tested in T-ALL clinical samples. By statistically comparing the expression results and finding reverse correlation between the expression of the genes and target miRNAs, it seems they can be used as effective biomarkers for diagnosis and as therapeutic targets after implementing necessary measures.
Conflict of interest
The authors confirm that they have no conflicts of interest on this article content.

Acknowledgments
The results described in this paper were part of Tohid Naderi’s thesis in Shahid Beheshti University of Medical Sciences. This project was funded by Shahid Beheshti University of Medical Sciences. The authors appreciate Pasteur Institute of Iran, Tehran, Iran, and also Cellular and Molecular Biology Research center, Shahid Beheshti University of Medical Sciences, Tehran, Iran for providing technical support.


References:
1.    Wang Z, Li Y, Ahmad A, Azmi AS, Banerjee S, Kong D, et al. Targeting Notch signaling pathway to overcome drug resistance for cancer therapy. Biochim Biophys Acta Rev Cancer. 2010;1806:258-267.
2.    Sarmento L, Póvoa V, Nascimento R, Real G, Antunes I, Martins L, et al. CHK1 overexpression in T-cell acute lymphoblastic leukemia is essential for proliferation and survival by preventing excessive replication stress. Oncogene. 2015;34:2978-2990.
3.    Lobry C, Oh P, Mansour MR, Look AT, Aifantis I. Notch signaling: switching an oncogene to a tumor suppressor. Blood. 2014;123:2451-2459.
4.    Hasserjian R, Aster J, Davi F, Weinberg D, Sklar J. Modulated expression of notch1 during thymocyte development. Blood. 1996;88:970-976.
5.    Liu J, Sato C, Cerletti M, Wagers A. Chapter twelve-notch signaling in the regulation of stem cell self-renewal and differentiation. Curr Top Dev Biol. 2010;92:367-409.
6.    Deftos ML, He Y-W, Ojala EW, Bevan MJ. Correlating notch signaling with thymocyte maturation. Immunity. 1998;9:777-786.
7.    van Limpt V, Chan A, Caron H, Sluis PV, Boon K, Hermus MC, et al. SAGE analysis of neuroblastoma reveals a high expression of the human homologue of the Drosophila Delta gene. Med Pediatr Oncol. 2000;35:554-558.
8.    Su Q, Xin L. Notch signaling in prostate cancer: refining a therapeutic opportunity. Histol Histopathol. 2016;31:149-157.
9.    Stylianou S, Clarke RB, Brennan K. Aberrant activation of notch signaling in human breast cancer. Cancer Res. 2006;66:1517-1525.
10.    Stockhausen MT, Kristoffersen K, Poulsen HS. The functional role of Notch signaling in human gliomas. Neuro Oncol. 2010;12:199-211.
11.    Ye Q, Jiang J, Zhan G, Yan W, Huang L, Hu Y, et al. Small molecule activation of NOTCH signaling inhibits acute myeloid leukemia. Sci Rep. 2016;6:26510.
12.    Malecki MJ, Sanchez-Irizarry C, Mitchell JL, Histen G, Xu ML, Aster JC, et al. Leukemia-associated mutations within the NOTCH1 heterodimerization domain fall into at least two distinct mechanistic classes. Mol Cell Biol. 2006;26:4642-4651.
13.    Joshi I, Minter LM, Telfer J, Demarest RM, Capobianco AJ, Aster JC, et al. Notch signaling mediates G 1/S cell-cycle progression in T cells via cyclin D3 and its dependent kinases. Blood. 2009;113:1689-1698.
14.    Gautschi O, Ratschiller D, Gugger M, Betticher DC, Heighway J. Cyclin D1 in non-small cell lung cancer: a key driver of malignant transformation. Lung cancer. 2007;55:1-14.
15.    Yang CC, Chu KC, Chen HY, Chen WC. Expression of p16 and cyclin D1 in bladder cancer and correlation in cancer progression. Urol Int. 2002;69:190-194.
16.    Bachmann K, Neumann A, Hinsch A, Nentwich MF, El Gammal AT, Vashist Y, et al. Cyclin D1 is a strong prognostic factor for survival in pancreatic cancer: analysis of CD G870A polymorphism, FISH and immunohistochemistry. J Surg Oncol. 2015;111:316-323.
17.    Cohen B, Shimizu M, Izrailit J, Ng NF, Buchman Y, Pan JG, et al. Cyclin D1 is a direct target of JAG1-mediated Notch signaling in breast cancer. Breast Cancer Res Treat. 2010;123:113-124.
18.    Ghildiyal M, Zamore PD. Small silencing RNAs: an expanding universe. Nat Rev Genet. 2009;10:94-108.
19.    Winter J, Jung S, Keller S, Gregory RI, Diederichs S. Many roads to maturity: microRNA biogenesis pathways and their regulation. Nat Cell Biol. 2009;11:228-234.
20.    Wang W, Luo Y-p. MicroRNAs in breast cancer: oncogene and tumor suppressors with clinical potential. J Zhejiang Univ Sci B. 2015;16:18-31.
21.    Abba ML, Patil N, Leupold JH, Moniuszko M, Utikal J, Niklinski J, et al. MicroRNAs as novel targets and tools in cancer therapy. Cancer lett. 2017;387:84-94.
22.    Karami F, Mohammadi-Yeganeh S, Abedi N, Koochaki A, Kia V, Paryan M. Bioinformatics Prediction and In Vitro Analysis Revealed That miR-17 Targets Cyclin D1 mRNA in Triple Negative Breast Cancer Cells. Chem Biol Drug Des. 2016;87:317-320.
23.    Mohammadi-Yeganeh S, Mansouri A, Paryan M. Targeting of miR9/NOTCH1 interaction reduces metastatic behavior in triple-negative breast cancer. Chem Biol Drug Des. 2015;86:1185-1191.
24.    Mohammadi-Yeganeh S, Paryan M, Samiee SM, Soleimani M, Arefian E, Azadmanesh K, et al. Development of a robust, low cost stem-loop real-time quantification PCR technique for miRNA expression analysis. Mol Biol Rep. 2013;40:3665-3674.
25.    Paryan M, Mohammadi-Yeganeh S, Samiee SM, Soleimani M, Arefian E, Azadmanesh K, et al. Investigation of deregulated genes of Notch signaling pathway in human T cell acute lymphoblastic leukemia cell lines and clinical samples. Mol Biol Rep. 2013;40:5531-5540.
26.    Li X, von Boehmer H. Notch signaling in T-cell development and T-ALL. ISRN Hematol. 2011;2011.
27.    Xu Y, Yang J, Li X. MicroRNA-25 promotes T-cell acute lymphoblastic leukemia cell proliferation and invasion by directly targeting EphA8. IJCEP. 2016;9:5292-5298.
28.    Shi C, Zhang X, Li X, Zhang L, Li L, Sun Z, et al. Effects of microRNA-21 on the biological functions of T-cell acute lymphoblastic lymphoma/leukemia. Oncol Lett. 2016;12:4173-4180.
29.    Cimmino A, Calin GA, Fabbri M, Iorio MV, Ferracin M, Shimizu M, et al. miR-15 and miR-16 induce apoptosis by targeting BCL2. Proc Natl Acad Sci U S A. 2005;102:13944-13949.
30.    Wang J, Chen J, Sen S. MicroRNA as biomarkers and diagnostics. J Cell Physiol. 2016;231:25-30.
31.    Mavrakis KJ, Van Der Meulen J, Wolfe AL, Liu X, Mets E, Taghon T, et al. A cooperative microRNA-tumor suppressor gene network in acute T-cell lymphoblastic leukemia (T-ALL). Nat Genet. 2011;43:673-678.
32.    Lv M, Zhang X, Jia H, Li D, Zhang B, Zhang H, et al. An oncogenic role of miR-142-3p in human T-cell acute lymphoblastic leukemia (T-ALL) by targeting glucocorticoid receptor-[alpha] and cAMP/PKA pathways. Leukemia. 2012;26:769-777.
33.    Gusscott S, Kuchenbauer F, Humphries RK, Weng AP. Notch-mediated repression of miR-223 contributes to IGF1R regulation in T-ALL. Leuk Res. 2012;36:905-911.
34.    Li X, Sanda T, Look AT, Novina CD, von Boehmer H. Repression of tumor suppressor miR-451 is essential for NOTCH1-induced oncogenesis in T-ALL. J Exp Med. 2011:663-675.
35.    Mohammadi Yeganeh S, Vasei M, Tavakoli R, Kia V, Paryan M. The effect of miR-340 over-expression on cell-cycle-related genes in triple-negative breast cancer cells. Eur J Cancer Care (Engl). 2017;26.
36.    Weng AP, Millholland JM, Yashiro-Ohtani Y, Arcangeli ML, Lau A, Wai C, et al. c-Myc is an important direct target of Notch1 in T-cell acute lymphoblastic leukemia/lymphoma Genes Dev. 2006;20:2096-2109.
37.    Palomero T, Lim WK, Odom DT, Sulis ML, Real PJ, Margolin A, et al. NOTCH1 directly regulates c-MYC and activates a feed-forward-loop transcriptional network promoting leukemic cell growth. Proc Natl Acad Sci U S A. 2006;103:18261-18266.
38.    Tao J, Zhao X, Tao J. c-MYC–miRNA circuitry: a central regulator of aggressive B-cell malignancies. Cell Cycle. 2014;13:191-198.
39.    Christoffersen N, Shalgi R, Frankel L, Leucci E, Lees M, Klausen M, et al. p53-independent upregulation of miR-34a during oncogene-induced senescence represses MYC. Cell death differ. 2010;17:236-245.
40.    Sun F, Fu H, Liu Q, Tie Y, Zhu J, Xing R, et al. Downregulation of CCND1 and CDK6 by miR‐34a induces cell cycle arrest. FEBS Lett. 2008;582:1564-1568.
41.    Du R, Sun W, Xia L, Zhao A, Yu Y, Zhao L, et al. Hypoxia-induced down-regulation of microRNA-34a promotes EMT by targeting the Notch signaling pathway in tubular epithelial cells. PloS one. 2012;7:e30771.
42.    De Weer A, Van der Meulen J, Rondou P, Taghon T, Konrad TA, De Preter K, et al. EVI1‐mediated down regulation of MIR449A is essential for the survival of EVI1 positive leukaemic cells. Br J Haematol. 2011;154:337-348.
43.    Capuano M, Iaffaldano L, Tinto N, Montanaro D, Capobianco V, Izzo V, et al. MicroRNA-449a overexpression, reduced NOTCH1 signals and scarce goblet cells characterize the small intestine of celiac patients. PloS one. 2011;6:e29094.
44.    Shi J, Liu Y, Liu J, Zhou J. Hsa-miR-449a genetic variant is associated with risk of gastric cancer in a Chinese population. Int J Clin Exp Pathol. 2015;8:13387-13392.
45.    Zhang C, Liu J, Tan C, Yue X, Zhao Y, Peng J, et al. microRNA-1827 represses MDM2 to positively regulate tumor suppressor p53 and suppress tumorigenesis. Oncotarget. 2016;7:8783-8796.
46.    Lin N, Zhou Y, Lian X, Tu Y. Expression of microRNA-106b and its clinical significance in cutaneous melanoma. Genet Mol Res. 2015;14:16379-16385.
47.    Jiang L, Li X, Cheng Q, Zhang B-H. Plasma microRNA might as a potential biomarker for hepatocellular carcinoma and chronic liver disease screening. Tumour Biol. 2015;36:7167-7174.